Overexpression of satellite RNAs in heterochromatin induces chromosomal instability and reflects drug sensitivity in mouse cancer cells

Overexpression of satellite RNAs in heterochromatin induces chromosomal instability (CIN) through the DNA damage response and cell cycle checkpoint activation. Although satellite RNAs may be therapeutic targets, the associated mechanisms underlying drug sensitivity are unknown. Here, we determined whether satellite RNAs reflect drug sensitivity to the topoisomerase I inhibitor camptothecin (CPT) via CIN induction. We constructed retroviral vectors expressing major satellite and control viruses, infected microsatellite stable mouse colon cancer cells (CT26) and MC38 cells harboring microsatellite instability, and assessed drug sensitivity after 48 h. Cells overexpressing satellite RNAs showed clear features of abnormal segregation, including micronuclei and anaphase bridging, and elevated levels of the DNA damage marker γH2AX relative to controls. Additionally, overexpression of satellite RNAs enhanced MC38 cell susceptibility to CPT [half-maximal inhibitory concentration: 0.814 μM (control) vs. 0.332 μM (MC38 cells with a major satellite), p = 0.003] but not that of CT26. These findings imply that MC38 cells, which are unlikely to harbor CIN, are more susceptible to CIN-induced CPT sensitivity than CT26 cells, which are characterized by CIN. Furthermore, CPT administration upregulated p53 levels but not those of p21, indicating that overexpression of major satellite transcripts likely induces CPT-responsive cell death rather than cellular senescence.

Many cancers exhibit "aneuploidy, " which is defined as the presence of an abnormal number of chromosomes in a cell 1 . Chromosomal instability (CIN) is characterized by a high frequency of chromosomal abnormalities, such as a gain or loss of entire chromosomes or large regions (aneuploidy), structural rearrangements, and localized abnormalities, such as amplifications and deletions 2,3 . It is estimated that 70-95% of cancers exhibit chromosomal abnormalities suggestive of CIN 4,5 . These alterations disrupt normal genome structure and function, increase the frequency of mutations, and cause epigenetic changes. Moreover, CIN increases diversity and heterogeneity, which in turn increases cancer malignancy and promotes drug resistance 6 .
The central part of the chromosome (the centromere) plays an important role in maintaining chromosomal stability, and its impairment facilitates abnormal segregation of chromosomes. The centromere comprises a 171-bp repetitive sequence called satellite DNA. Satellite α transcripts (SATs) are a heterogeneous population of noncoding RNAs transcribed from satellite DNA and include large swaths of repetitive sequences at the centromere and telomeres of a variety of eukaryotic chromosomes [7][8][9] . A landmark study in fission yeast demonstrated that the transcription of satellite RNAs is critical for the establishment and maintenance of pericentromeric heterochromatin 10 . Heterochromatic repetitive satellite RNAs are extensively transcribed in various human cancers 11 . Additionally, aberrant expression of satellite RNAs in cultured cells induces a DNA damage response, www.nature.com/scientificreports/ activates cell cycle checkpoints, and causes defects in chromosome segregation 12,13 . Furthermore, we have previously demonstrated that overexpression of SATs induced by retroviral expression vectors leads to changes in copy number at specific chromosomes 14 . CIN is strongly associated with drug resistance, and numerous reports have indicated that CIN correlates with resistance to antineoplastic agents, such as taxol, in both tumor-derived cell lines and clinical settings 6,[15][16][17] . However, excessive levels of CIN reportedly increase sensitivity to cytotoxic therapies, such as cisplatin and 5-fluorouracil (5-FU), in ovarian, rectal, and breast cancers [17][18][19][20] . A previous study showed that inducing wholechromosome missegregation sensitizes transplanted human glioblastoma tumors to radiation treatment 21 . Furthermore, misexpression of genes at the centromere and kinetochore regions is reportedly associated with outcomes in cancer patients and their response to radiotherapy and chemotherapy 22 . Drug-induced genotoxicity leads to CIN, which may reduce tolerance to genotoxic stress in cancer cells. For example, replication forks are often stalled owing to replication stress, which causes genomic instability. Additionally, overexpression of satellite RNAs decreases the expression levels of proteins that play a role in stabilizing and repairing stalled replication forks 13 . Moreover, the formation of RNA-DNA hybrids at the replication fork prevents the re-stalling of replication forks. In cancer treatment, these types of genetic stress represent ideal targets for mediating cancer cell death.
DNA topoisomerase I inhibitors prevent the repair of single-stranded DNA breaks, leading to cancer cell death. Topoisomerase I inhibitors, such as irinotecan and topotecan, are camptothecin (CPT) analogs that damage DNA 23 . Topoisomerase I normally forms a DNA-topoisomerase I complex during DNA replication and translation, which relaxes the DNA double helix structure by creating a single-stranded DNA state and recombining the DNA. Topoisomerase inhibitors inhibit DNA recombination by stabilizing the DNA-topoisomerase I complex, ultimately leading to double-strand breaks and cell death. Zhang et al. 22 reported that cancer cell lines with high gene expression at the centromere and kinetochore regions are more sensitive to topoisomerase I inhibitors than those with a low expression, and that genotoxicity decreases the survival of cells with CIN by reducing their tolerance to genotoxic stress. In this study, we determined whether satellite RNA-induced CIN enhances the sensitivity to topoisomerase I inhibitors, which prevent the repair of DNA single-strand breaks.

Results
Overexpression of satellite RNA and its effects on cell growth of cancer cell lines. To elucidate the effects of the overexpression of major satellite RNAs (mSATs) in cancer cell lines, we transduced mSATs using retroviral vectors into CT26 and MC38 cell lines. CT26 is a microsatellite stable (MSS) cancer cell line, whereas MC38 is a cell line with microsatellite instability (MSI). Successful transfection induced mSAT overexpression in these cells, with the expression levels of major satellites confirmed by qRT-PCR. The results verified the higher expression of mSATs in cells infected with major satellite-positive viruses relative to the control vector (control, 22.66 ± 11.24% vs. mSAT, 101.66 ± 4.41% in CT26 cells; 27.0 ± 1.15% vs. 90.66 ± 5.20% in MC38 cells; *p < 0.05, **p < 0.01, respectively) (Fig. 1a). We then investigated the effects of mSAT overexpression on cell growth and compared the number of mSAT-transfected and control CT 26 and MC38 cells after 4 days of culture. We observed gradual cell growth in both cell lines, although no difference in cell growth was observed between mSAT-transfected and control cells (Fig. 1b), as confirmed by soft agar colony formation assay (Fig. 1c).
We evaluated the DNA damage response based on the number of anti-γH2AX-positive cells and found that the proportion of anti-γH2AX-positive cells significantly increased in mSAT-overexpressing CT26 and MC38 cells compared with the control cells (control: 3.99 ± 0.13% vs. 36.26 ± 0.67% in CT26 cells; 6.78 ± 0.16% vs. 38.17 ± 2.77% in CT26 cells; *p < 0.05 and **p < 0.01, respectively, Fig. 3a). Additionally, Fig. 3b shows the images representing the evaluation of the incidence of anti-γH2AX-positive mSAT-overexpressing CT26 cells in three field of view per sample (representative of three independent experiments).
Overexpression of satellite RNAs reflects drug sensitivity. We determined drug sensitivity at 48 h after treatment with several doses of CPT and found that the growth of CT26 and MC 38 cells is inhibited in a dose-dependent manner. Notably, overexpression of mSAT increased the drug sensitivity of MC38 cells but not that of CT26 cells to CPT (Fig. 4a,b). In particular, MC38 sensitivity to CPT was significantly enhanced (IC 50 : control, 0.814 μM vs. mSAT, 0.332 μM in MC38 cells, p = 0.003), whereas that of CT26 cells had no significant difference (IC 50 : control, 0.260 μM vs. mSAT, 0.256 μM, p = 0.955). These findings imply that MC38 cells, which are unlikely to harbor CIN, are more susceptible to CIN-induced CPT sensitivity than CT26 cells, which are characterized by CIN.
We also determined drug sensitivity after treatment with oxaliplatin, a platinum anticancer agent. Both CT26 and MC 38 cells had inhibited growth in a dose-dependent manner; however, they showed no significant

CPT treatment alters levels of apoptosis-related proteins in MC38 cells.
We performed immunoblot analysis to verify the changes in the levels of apoptosis-related proteins and those associated with cellular senescence, such as p53 and p21. Treatment of MC38 cells with and without mSAT overexpression with 5 μM CPT for 2 h, 4 h, and 8 h resulted in significant increases in p53 levels in MC38 cells overexpressing mSAT in a time-dependent manner, whereas these changes were not observed in the control (Fig. 5a). Additionally, we observed significant increases in p21 levels in a time-dependent manner regardless of mSAT-expression status in MC38 cells, suggesting that mSAT overexpression induces cell death rather than cellular senescence in response to CPT treatment (Fig. 5a). Regarding the levels of apoptosis-related proteins bcl-2 and cleaved caspase-3, bcl-2 was not expressed, whereas caspase-3 was expressed in a time-dependent manner; however, no significant difference was observed between the control and mSAT cells (Fig. 5b). Figure 5c shows the relative intensities of protein expression as determined by Image J.

Discussion
In this study, we demonstrated that mSAT overexpression induces CIN and DNA damage and increases the drug sensitivity of the mouse colon cancer cell line MC38 (MSI) to the topoisomerase I inhibitor CPT. Another mouse colon cancer cell line, CT26 (MSS), did not show a change in CPT sensitivity. These findings may imply that MC38 cells, which are unlikely to harbor CIN, are more susceptible to CIN-induced CPT sensitivity than CT26 cells, which are characterized by CIN. Furthermore, we observed increases in p53 levels but not in those of p21 in CPT-treated MC38 cells, suggesting that mSAT overexpression likely induces cell death rather than cellular senescence in response to CPT. We found that mitotic errors, including abnormal segregations, micronuclei formation, and anaphase bridging, are enhanced by mSAT overexpression, followed by DNA damage in mouse cell lines, demonstrating that mSAT overexpression induces CIN. Zhu et al. 13 reported that CRISPR-mediated activation of satellite RNA www.nature.com/scientificreports/ expression induces CIN in breast cancer cell lines, and Kishikawa et al. 24,25 showed that mSATs cause chromosomal and genomic instability in a mouse pancreatic cancer cell line. Additionally, we have previously demonstrated that retrovirus-mediated SATs lead to changes in copy number at specific chromosomes in normal human mammary epithelial cells 14 . Overexpression of satellite RNAs is reportedly upregulated in various types of cancers, including those in humans and mice 11 , implying that satellite RNAs that induce CIN are involved in the development of some types of cancer through genomic diversity and heterogeneity 6 .
Multiple studies have reported that drug resistance is induced by CIN 6,15-17 ; however, in this study, we found that drug sensitivity is enhanced by treatment with the topoisomerase I inhibitor CPT. This finding is consistent with the increased effect of CPT in cancer cell lines described by Zhang et al. 22 , where CIN reduced the tolerance to genotoxic stress in response to CPT. Additionally, Swanton et al. 17 demonstrated that sensitivity to carboplatin (a platinum anticancer agent) is increased by CIN but had a reverse effect to taxane (a microtubule-stabilizing inhibitor) in patients with ovarian cancer. CIN is functionally associated with altered intrinsic tumor sensitivity to two distinct drug agents. In this study, drug sensitivity to oxaliplatin (a platinum anticancer agent) was not changed by overexpression of satellite RNAs in mouse colon cell lines, suggesting that CIN-induced drug sensitivity depends on the types of tumor cells. Zhu et al. 13 demonstrated that satellite RNA-overexpressing cells are more sensitive to DNA-replication stress than DNA double-strand breaks, which is related to our finding that induction of CIN in satellite RNA-overexpressing cells is more sensitive to the DNA-replication stress induced by CPT than the DNA crosslink effects of oxaliplatin. Jamal et al. 18 showed that extreme CIN predicts improve outcomes in patients with breast cancer, although the mechanisms underlying the induction of drug sensitivity to a specific agent by CIN were not identified. Zaki et al. 20 reported that widespread DNA damage resulting from lagging chromosomes and chromatin bridges sensitizes tumor cells to additional damage caused by ionizing radiation in combination with 5-FU. Imbalanced genetic stress induced by CIN could contribute to the sensitivity or resistance to each drug.
Disruption of the cell cycle induces cell death, senescence, and altered proliferation. In this study, MC38 cells overexpressing mSAT did not have altered proliferation but had increased cell death, along with increased www.nature.com/scientificreports/ p53 levels, in response to CPT treatment. Aurora kinase A (AURKA), a cell cycle-regulated kinase involved in spindle formation and chromosome segregation 26 , is associated with CIN in colorectal cancer 27 and multi-drug resistance in breast cancer 28 . In this study, AURKA levels increased regardless of the presence or absence of mSAT overexpression in response to CPT ( Supplementary Fig. 2). Innate immune signaling can be activated by errors in chromosome segregation and replication stress through the introduction of genomic double-stranded DNA (dsDNA) into the cytosol and engagement of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) cytosolic dsDNA-sensing antiviral pathway [29][30][31][32] . The consequences of CIN not only involve tumor-cell autonomy but also the cross-talk between cancer cells and their immune microenvironment 33 . Although we expected to observe activation of the cGAS-STING pathway by mitotic errors induced by mSAT overexpression, we found that cGAS is expressed before CPT treatment, and that cGAS expression does not increase over time. In contrast, STING was barely expressed in control cells over time but was expressed in mSAT cells before CPT treatment, suggesting that STING might be triggered by mitotic errors induced by mSAT overexpression (Supplementary Fig. 2).
This study has some limitations. First, we used only two colon cancer cell lines as representatives of MSI and MSS cancer; therefore, an in vivo study is required to confirm our in vitro findings. Second, clinical investigations should be conducted in patients with colon cancer treated with CPT by comparing tumor specimens between patients with high expression of human SATs and those with a low expression.
In summary, we demonstrated that the overexpression of a mouse major satellite induces CIN and enhanced drug sensitivity to the topoisomerase I inhibitor CPT in MC38 cells, which are unlikely to harbor CIN and intrinsically resistant to CPT than CT26 cells, which are characterized by CIN. The intrinsic feature of drug resistance to CPT in MC38 cells could be overcome to some extent by satellite RNA overexpression. Our findings offer insight into the different aspects of CIN associated with drug treatment in patients with colon cancer.   Memorial Institute-1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 mL streptomycin. All cells were cultured at 37 °C in a 5% CO 2 atmosphere. The plasmid vector p156RRL-EF1a-GFPU3H1MajSat (#41796; Addgene, Watertown, MA, USA) was a gift from Inder Verma and has been previously described 13 .

Methods
To investigate whether the overexpression of major satellite RNA reflects drug sensitivity, we constructed retroviral control vectors and vectors expressing mSAT (Supplementary Fig. 1). cDNA of elongation factor 1α (EF1α), the SV40 polyA signal, and major satellites were amplified using polymerase chain reaction (PCR) with PrimeSTAR GXL DNA polymerase (Takara Bio, Shiga, Japan) and inserted into the EcoRI and SalI sites of the MSCV-Puro retroviral vector. To prepare the control vector, only EF1α and the SV40 polyA signal were inserted into the said sites of the MCSV puroviral retroviral vector.
To obtain retroviruses, retroviral vectors expressing mSATs and control sequences were transfected into HEK293T cells (1 × 10 6 cells/60-mm-diameter culture dish), along with helpers, such as pE-Eco and pGP (Takara Bio). After 24 h, the culture medium was replaced with 1.5 mL of fresh culture medium. Secreted retroviruses were harvested every 4 h during the 24-to 60-h post-transfection period, pooled, and stored on ice. Exponentially growing cells (1 × 10 5 cells/60-mm-diameter culture dish) were infected with 2 mL of virus-containing    Western blot analysis. Cells were lysed with radioimmunoprecipitation assay buffer without sodium dodecyl sulfate (SDS) [10 mM sodium phosphate (pH 7.2), 150 mM NaCl, 3 mM MgCl 2 , 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.2 U/mL aprotinin, and phosphatase inhibitors] and briefly sonicated on ice. Debris were removed by sedimentation in a microcentrifuge at 16,400 × g for 10 min at 4 °C, and the cleared cell lysates were harvested and mixed with Laemmli sample buffer. Proteins (25 µg) from whole-cell lysates were loaded in each lane of an SDS-polyacrylamide gel, separated by electrophoresis, transferred onto polyvinylidene difluoride membranes (Merck Millipore), and visualized by immunoblotting with the indicated antibodies and enhanced chemiluminescence (GE Healthcare, Provo, UT, USA). The relative intensities of protein expression were determined by Image J (http:// rsb. info. nih. gov/ ij/ index. html). The relative ratio was calculated by comparing with the amount before CPT treatment. The ratio before CPT treatments was set as 1 in both the control and mSAT cells.
Statistical analyses. All statistical analyses were performed using GraphPad Prism (v.9.0; GraphPad Software, San Diego, CA, USA). When necessary, differences in qualitative variables were evaluated using either the χ 2 test or Fisher's exact test. Continuous variables were compared using analysis of variance with the Tukey-Kramer test, and the means or medians were compared with the paired samples t-test for normally distributed variables. Dose-response curves with the responses normalized to the zero dose as a function of log concentration were generated and statistically compared using the sum-of-squares F test. The half-maximal inhibitory concentration (IC 50 ) values were obtained using a four-parameter logistic model. Statistica signifcance was set at p < 0.05.

Data availability
The datasets that support the findings of this study are available from the corresponding author on reasonable request.